Exosome Release Delays Senescence by Disposing of Obsolete Biomolecules

Abstract Accumulation of obsolete biomolecules can accelerate cell senescence and organism aging. The two efficient intracellular systems, namely the ubiquitin‐proteasome system and the autophagy‐lysosome system, play important roles in dealing with cellular wastes. However, how multicellular organisms orchestrate the processing of obsolete molecules and delay aging remains unclear. Herein, it is shown that prevention of exosome release by GW4869 or Rab27a −/− accelerated senescence in various cells and mice, while stimulating exosome release by nutrient restriction delays aging. Interestingly, exosomes isolate from serum‐deprived cells or diet‐restricted human plasma, enriched with garbage biomolecules, including misfolded proteins, oxidized lipids, and proteins. These cellular wastes can be englobed by macrophages, eventually, for disintegration in vivo. Inhibition of nutrient‐sensing mTORC1 signaling increases exosome release and delays senescence, while constitutive activation of mTORC1 reduces exosome secretion and exacerbates senescence in vitro and in mice. Notably, inhibition of exosome release attenuates nutrient restriction‐ or rapamycin‐delayed senescence, supporting a key role for exosome secretion in this process. This study reveals a potential mechanism by which stimulated exosome release delays aging in multicellular organisms, by orchestrating the harmful biomolecules disposal via exosomes and macrophages.

amplification primers were designed according to the KO sequence, and the genotypes of the F0 generation mice were identified. Rab27a KO mice had grayer coats than wild-type mice. All lines were backcrossed to the C57BL/6J background. C57BL/6 mice (newborn or 6-7 weeks old) were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, China). The exosome inhibitor GW4869 (Sigma-Aldrich) was injected into the tail vein after placing the mouse in a fixer, straightening the tail, and wiping it with an alcohol cotton ball. The tail was then held with the left hand and the syringe with the right hand. The needle was inserted slowly until the blood returned, and the solution was then injected slowly.
GW4869 was dissolved in dimethylsulfoxide (DMSO) at 8 mg/mL. The working solution was freshly prepared before use in 0.9% normal saline, with a final concentration of 0.3 mg/mL (2.5 µg/g body weight). For intermittent dietary restriction in elderly mice (14 months old), the mice were allowed to drink and eat freely every other day for 5 months. Mice were bred and maintained under specific pathogen-free conditions in a temperature-and humidity-controlled environment with a 12 h light/dark cycle (06:00-18:00).

Isolation of primary cells
Isolation of hUCB-MSCs: hUCB-MSCs were isolated from newborn placenta according to a previously described method (Kern et al., 2006). Briefly, prior to the isolation of multinucleated cells, anticoagulated cord blood was diluted 1:1 with 2 mM EDTA-PBS. The isolated cells were then seeded at a density 2×10 5 cells per cm 2 on plates in growth medium containing D-media (Formula 78-5470EF; Gibco, Carlsbad, CA, USA), the endothelial cell growth medium-2 (EGM-2), and 10% FBS (Gibco). Adherent fibroblastoid cells which appeared as colony forming unit-fibroblastic (CFU-F) were then harvested at subconfluence using trypsin (Gibco) and sub-cultured (1).
Differentiating macrophages from bone marrow cells: Mice were euthanized by CO 2 inhalation, and death was ensured by cervical dislocation. The femur was separated from the tibia by dislocation and cutting through the knee joint, the tibia was separated from the foot by cutting just below the ankle joint, and tissue was removed from the bones. The epiphyses of the bones were cut and the marrow was flushed into a 50 mL centrifuge tube using a 5 mL syringe and a 23 G needle, with 5 mL of phosphate-buffered saline (PBS) per bone. The cell suspension was then filtered through a 70 μm cell strainer into a new 50 mL tube and centrifuged for 5 min at 250 × g and 4°C. The supernatant was discarded and the cell pellet was resuspended in 3 mL of bone marrow-derived macrophages (BMDM) medium. Twenty microliters of the suspension was diluted in 180 μL PBS, and 20 μL of this dilution was added to 80 μL of 2% acetic acid, and incubated for 2-3 min at room temperature. The cells were counted under a microscope using a hemocytometer. Seven milliliters of BMDM medium was then added to the non-diluted cells and 10 mL of suspension was seeded on a 10 cm cell culture-treated petri dish and incubated for 3 h at 37°C with 5% CO 2 .
The cells were then diluted to a concentration of 3.5×10 5 cells/mL, macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA) was added at a final concentration of 25 ng/mL, and 10 mL of cells was seeded per 10 cm cell culture-treated petri dish and incubated at 37°C with 5% CO 2 for 3 days. The medium was then replaced for further incubation(2).
Isolation of MEFs from mouse embryos: Pregnant female mice were anesthetized at day E14.5 by intraperitoneal injection of 0.5 mL 2.5% avertin, and then euthanized by cervical dislocation. A small incision was cut through the skin at the centric position on the middle abdomen. The embryos were removed and placed in a sterile dish, and 3 mL of ice-cold 0.25% trypsin-EDTA was added to the dish. Two pairs of fine forceps were then used to tease the fetuses into fine pieces. All the material was transferred to a 15-mL tube (for up to four embryos) using a pipette, and ice-cold 0.25% trypsin-EDTA was added to bring the total volume to 3 mL per embryo. The tube was allowed to stand overnight at 4°C, and was then incubated for 30 min in a 37°C water bath. MEF culture medium was added to 8 mL (per 15-mL tube) and pipetted vigorously and repeatedly up and down to break up the digested tissues into a cell suspension. The cell suspension was allowed to settle for 1 min to let any remaining clumps of tissue fall to the bottom of the tube. The supernatant cell suspension was then transferred to a new tube and mixed, and the cells were plated in 10-cm tissue culture dishes with 10 mL MEF culture medium(3).

Cell culture
MEFs and HUVECs were cultured in high-sugar Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% FBS. hUCB-MSCs were inoculated into 10% FBS, 1% sodium pyruvate, L-glutamine, MEM non-essential amino acids, low-sugar DMEM medium (all from Gibco) and placed in an incubator, and subcultured at ratios of 1:3 or 1:4. GW4869 powder 2 mM was dissolved in DMSO (Sigma-Aldrich) and sterilized by a 220-nm filter. Rapamycin (MCE, Monmouth Junction, NJ, USA) was dissolved in DMSO (Sigma-Aldrich), sterilized by a 220-nm filter, and 100 nM was added to the culture medium. All cells were cultured in a 37°C incubator with 5% CO 2 and 95% humidity. The culture medium was replaced every other day, or according to experimental requirements.

Isolation of exosomes
The culture medium (depleted from serum exosomes) was collected and centrifuged at 320 × g for 10 min to pellet the cells, followed by 2,000 × g for 10 min to remove cell debris and dead cells, and 10,000 × g for 30 min at 4°C. The medium was further centrifuged at 100,000 × g for 70 min at 4°C (Beckman Coulter Optima L-100 XP, Beckman Coulter). The precipitate was washed twice with PBS, centrifuged at 100,000 × g for 70 min, and the exosomes at the bottom of the tube were collected.
Whole blood was drawn into an EDTA anticoagulation tube and centrifuged to obtain separated plasma. Samples were centrifuged at 3000 × g for 15 min at 4°C and transferred to a new 1.5 mL centrifuge tube, followed by centrifuging at 10,000 × g for 30 min at 4°C. The supernatant was then diluted with PBS buffer and transferred to a Pollyallomer high-speed centrifuge tube (Sigma-Aldrich) and centrifuged at 100,000 × g for 70 min at 4°C using a ultracentrifuge (Beckman). The pellet was washed twice with PBS and centrifuged at 100,000 × g for 70 min to collect the exosomes at the bottom of the tube, before resuspension in RIPA buffer (Sigma-Aldrich). Protein concentration was determined by BCA Protein Assay (Abcam, Cambridge, UK).
Following centrifugation, 1 mL fractions were collected from top to bottom, centrifuged at 100,000 × g for 1 h at 4°C, and resuspended in 50-80 μL PBS.

Nanosight analysis
The exosomal samples were diluted and the concentration was adjusted as required (1 × 10 8 -1 × 10 11 /mL) for characterization. The number and size distribution of the exosomes were detected using a Nanosight N3000 nanoparticle analyzer (Malvern, UK), and the data were analyzed using the related software NTA 3.2 Dev Build 3.2.16 (Malvern).

SA-β-gal assay
Senescent cells were stained using an SA-β-gal staining kit (Cell Signaling Technology, Cat#9860S, Boston, MA, USA). MEFs, HUVECs, and hUCB-MSCs in 12-well plates, and frozen tissue section were fixed with 4% paraformaldehyde for 10 min at room temperature and stained overnight at 37°C. The staining solution was then removed and the samples were washed in PBS. Senescent cells, identified as blue-stained cells, were detected by light microscopy (Nikon TE2000-U; Zeiss Axio Scope.A1, Carl Zeiss, Oberkohen, baden-Wurberg, Germany).
Frozen tissue sections were detected using an SA-β-gal staining kit (Beyotime, Jiangsu, China). Mice were euthanized and tissues were fixed in 4% paraformaldehyde for 24 h, followed by transfer to 30% sucrose for 24 h for dehydration. Sagittal sections (5 μm) were prepared using a Leica CM1850 cryostat (Leica, Wetzlar, Germany) and incubated for 15 min at 37°C to remove opti-mum cutting temperature compound (OCT) embedding medium, and then stained using SA-β-gal staining.

Immunohistochemistry
Immunohistochemistry was performed on 4 μm sections of formalin-fixed paraffin-embedded tissues. The sections were dehydrated and deparaffinized using xylene and graded ethanols, and then soaked in citrate buffer (10 mM citric acid, pH6.0) for 16 h at 60°C to unmask antigens. After rewarming and washing with PBS, 3% hydrogen peroxide solution was added dropwise and incubated at room temperature for 10 min. The sections were then blocked with 1% goat serum at 37°C Stained sections were imaged on an Axio Scope A1 microscope (Carl Zeiss).

Real-time RT-PCR
Cells were collected and washed twice with cold PBS (pH7.4). Total RNA was extracted using Trizol plus RNA purification kit (Takara Bio, Japan). Total RNA (0.2-1 μg) was reverse transcribed into cDNA using a PrimeScript RT kit (Vazyme Biotech, Nanjing, China) and RT-PCR was carried out using SYBR Premix Ex Taq II (Vazyme Biotech). Relative quantification was calculated using the 2 −ΔΔct method. All data were normalized against endogenous GAPDH controls of each sample.

Cell Counting Kit-8 (CCK-8) assay
The CCK-8 assay (MCE, Monmouth Junction, NJ, USA) was used to determine cell viability. A total of 2×10 3 cells (100 μL/well) were seeded in 96-well plates and allowed to adhere for 24 h, followed by the addition of 10 μL of CCK-8 solution to each well (being careful not to generate air bubbles). The cells were incubated for 2 h at 37°C and the absorbance was measured at 450 nm using a microplate reader (Synergy HTX Multi-Mode Reader, BioTek, Santa Clara, CA, USA) .

In vivo imaging and tissue distribution
Plasma was obtained from fasting mice and the exosomes were extracted by ultracentrifugation and incubated with DiD (a hydrophobic dye; AAT Bioquest, CA, USA) at room temperature for 10 min. The exosomes (50 μg/mL) were then injected into the tail vein of mice. The mice were anesthetized with pentobarbital (35-40 mg/kg). The comatose mice were shaved and the distribution of the exosomes was investigated using a near-infrared in vivo imaging system (FX Pro, Bruker, Madison, WI, USA). After 6 h, the mice were euthanized and their organs were imaged and tested.

Zetasizer nanoanalysis
Protein samples were lysed in 100 μL RIPA buffer (Sigma-Aldrich). The granularity of the particles was measured using a nanosizer (Malvern, UK), according to the manufacturer's instructions, with a conductivity value of 0.093 mS/cm and a voltage of 3.9 V.W.

Circular dichroism
Exosomes or cell suspension 100 μL of sonication (50 w 1s on, 2 s off, 2 min). After sonication, centrifuge at 12,000 × g, 10 min, 4°C, remove the supernatant and dilute to 300 µL with PBS. The circular dichroism instrument (Chirascan, Applied Photophysics Ltd, UK) was turned on and nitrogen gas was purged for at least 20 min.
Set parameters to temperature 4°C; wavelength 180-260 nm; then add 300 µL of sample to the cuvette for detection.

Quantification and statistical analysis
Quantitation was performed using Image J software (NIH, Bethesda, MD, USA).
Statistical analyses were carried out using GraphPad Prism 6.0 software (GraphPad Prism, San Diego, CA, USA). Differences between two groups were analyzed by two-tailed unpaired Student's t-test. A p value < 0.05 was considered statistically significant. All experiments were repeated at least three times. Quantitative data were expressed as mean ± standard deviation. Statistical analysis used in each panel was described in the figure legends.      were sacrificed and tissues were fixed and stained with the anti-pS6, anti-γH2AX and anti-lamin B1 antibodies, respectively, for immunohistochemical analysis. Scale bars = 50 μm.

Materials and Resources
Relative positive cells were quantified. *p < 0.05. and Lamin B1 mRNA were determined by qRT-PCR. GAPDH was used as the internal control.